Introduction  

Liver is the most important organ in the body that carries out vital physiological functions including metabolism of essential nutrients (carbohydrates, proteins, and lipids), energy utilization, metabolism and detoxification of various xenobiotics and medications, and excretion of waste substances [1, 2]. Liver metabolizes xenobiotics that lead to drug-induced liver injury (DILI) and is a possible complication of various therapeutic medications [3]. DILI is usually classified into two types: idiosyncratic and intrinsic; idiosyncratic DILI is and mostly dose-independent unpredictable while intrinsic DILI is predictable and dose-dependent (eg: toxicity of acetaminophen) [4]. DILI is one of the main reasons for the withdrawal of many drugs; Mechanism of hepatotoxicity could be mediated through mitochondrial dysfunction and DNA damage; such dysfunction could be caused by the drug itself or its cytochrome P450 mediated metabolite [5]. Various factors can attribute to hepatotoxicity like: genetics, environmental, gender variation, some drugs (eg; anticancer, antiviral, acetaminophen and some NSAIDs, and anti-mycobacterial), and certain disorders (eg; TB, HIV, and hepatitis) [6].

Methotrexate is a folic acid antagonist that is, extensively used as a chemotherapeutic agent to treat different cancerous stages such as acute lymphoblastic leukemia and other autoimmune inflammatory disorders (eg: multiple sclerosis, dermatomyositis, psoriasis, and rheumatoid arthritis) [7]. MTX is associated with many side effects including; gastrointestinal, hepatic and renal dysfunction, hematologic side effects, central and peripheral nervous system toxicity as well as respiratory toxicity [8]. Both high and low-dose therapy can lead to hepatotoxicity. Methotrexate at a very low dosage can affect the liver and cause changes in liver histology. Also, prolonged use or high-dose methotrexate (as in leukemia) can cause hepatotoxicity that may result in progressive fibrosis and cirrhosis. Hepatotoxicity resulting from the long-term use of methotrexate, will always remain a constant restriction of using this medication in the desired doses. It seems inhibiting the formation of tetrahydrofolate is responsible for both the toxic and therapeutic effects of methotrexate [9].

Olea europaea L., also known as Olive, belongs to the family Oleaceae which includes about 30 genera. Olive trees are native in temperate, tropical, and subtropical countries worldwide; and best known in Asia and Malaysia, especially the temperate and tropical regions of Asia [10]. Olive trees are distributed normally in the coastal regions of southeastern Europe and the eastern Mediterranean region, northern Africa, western Asia, and northern Iran at the south end of the Caspian Sea. The olive tree and fruit are important in the religion, and they have been mentioned in the Holy Quran and Bible [11]. O. europaea has some traditional medicinal uses; it is extensively used for various ailments in different countries. For example, olive leaves extract has cardioprotective and hypotensive effects; olive oil can be used orally as a laxative or applied externally for inflammation; and sometimes the oil can be used with lemon juice to treat urinary tract infection [12]. The extracts of the fruit and leaves are orally used as mouth cleanser, as well as to treat some intestinal and stomach discomfort, urinary tract and respiratory infections, and diarrhea. Continuous use of olive oil to the scalp can also usefully prevent hair loss. Also, different parts of olive trees possess anticarcinogenic, anti-oxidant, anti-inflammatory, anti-diabetic, and neuroprotective activities [13]. Phytochemical analysis carried out on different parts of O. europaea using various analytical methods like; HPLC, HPLC-MS, LC-MS, GC-MS, etc. revealed that they possess phenolic compounds, flavonoids, secoiridoids, secoiridoid glycosides, benzoic acid derivatives, sterols and triterpenes [14].

This study aimed to determine the hepatoprotective effect of different extracts of Olea europaea L. seeds in mice induced with liver injury by MTX and find out the most effective extract. 

Materials and Methods

Materials and chemicals

Olive seeds (Olea europaea L.) were obtained from the local market (Baghdad, Iraq) and their authenticity was confirmed by the Department of Pharmacognosy, College of Pharmacy, Al-Mustansiriyah University. Methotrexate (MTX) vials for injection (5mg/2ml) were obtained commercially as MTX vial from Mylan pharmaceuticals (UK). Silymarin was obtained commercially as Legalon^® capsules 135mg from Madaus pharmaceuticals (Greece). Ascorbic acid was obtained as powder for analytical purposes from Romil (UK). DPPH was obtained commercially as 99% DPPH powder for analysis produced in the USA.

Extraction process

Olive seeds were cleansed from fruit flesh, washed with tap water, and left to dry. Then, they were dried in an over at 40 °C for 0.5h then left to cool down, grounded into a fine with a final weight of 92 g. Olea europaea seeds powder was extracted with three solvents with varying polarities in a sequential manner (chloroform, methanol, and 50% aqueous ethanol), starting with low polar solvent and ending with the highest polarity using the cold method (maceration). The powder was soaked with each solvent at a ratio of (1:4 W/V) and left in a shaking water bath for 24h at 40 °C. Then it was filtered using Whatman filter paper no. 1for 3 times, concentrated using the rotary evaporator (Buchi, Switzerland), and put in a freeze dryer to obtain the lyophilized powder. The yield (%) for each extract was then calculated [15].

Experimental animals and grouping

The study was conducted in Pharmacology and Toxicology Lab. / Al-Rasheed University College / Pharmacy Department, Baghdad; Iraq after obtaining the ethical committee approval. All experiments were performed according to the National Institute of Health Guidelines for the care and use of laboratory animals and the European Council Directive on 24 November 1986 for Care and Use of Laboratory Animals (86/609/EEC), and approved by the Local Ethics Committee. 48 male albino mice aged 8 – 10 weeks old with a mean weight of (32.5 + 2 gm) were bought from the National Center for Drug Control and Research, Ministry of Health, Baghdad; Iraq. The mice were divided into 6 groups each with 8 mice (N = 8) housed under specific pathogen-free conditions at 23 + 2 °C and 12 hr light/dark cycle, and left to acclimatize for at least 1 week. They were fed with water and formal forage ad libitum in the animal house of Al-Rasheed University College, Pharmacy Department, Baghdad; Iraq. Group (1) represents the apparently normal animals and were considered as the reference group; Group (2) represents the liver injury-induced animals in which methotrexate (MTX) was given as a single dose (20mg/kg) / IP [16], Group (3) represent the animals treated with standard silymarin at a dose of (200mg/kg per day) and a single dose of MTX [17], Groups (4, 5, and 6) represents animals treated with Olea europaea chloroform (OE-CE), methanol (OE-ME) and 50% ethanol (OE-50%EE) extracts respectively with a daily dose of (500mg/kg) and a single dose of MTX [18]. All animal groups except the normal group received a single dose of MTX (20mg/kg/IP) on day 5 of the experiment; and all animal groups except for the normal group received the test substances at their mentioned respective doses starting from day 1 till day 5 of the experiment through oral gavage. On day 6 of the experiment, mice were sacrificed under anesthesia and samples were collected for further analysis.

Biochemical analysis

At the end of the experiment (day 6), mice were put under anesthesia with diethyl ether, blood was collected through heart puncture in plain tubes and then the samples were put in a centrifuge (LabTech, Korea) adjusted to 5000 rpm for 10 minutes to separate plasma. Plasma was then collected in separate tubes to be used in the biochemical analysis required to assess liver indices including; serum AST (GOT), ALT (GPT), ALP, total serum protein (TSP), and total serum bilirubin (TSB). The analysis was carried out using an automation analyzer according to the manufacturers kit.

Histological examination

Liver tissues were collected from all animal groups, fixed in 10% formalin for 48 h, and then processed to obtain paraffin-embedded liver tissue. Paraffin-embedded tissues were sectioned later on into layers with 5 µm thickness, which was then used to make the histological slide. Finally, the histological section was stained with hematoxylin & eosin stains and the section was viewed under a light microscope with 40X.

Free radical scavenging activity by (DPPH) assay

DPPH method was selected to measure the free radical scavenging activity of Olea europaea seeds extracts. A stock solution of 0.1mM DPPH was prepared by dissolving (0.04mg/ml) with absolute methanol. The extracts of Olea europaea seeds were tested at six different concentrations (500, 250, 125, 62.5, 31.25, & 15.63 µg/ml) by diluting each concentration to half; the procedure was carried out in a 96-well plate in which 100 µl of each concentration of each test extract was mixed with 200 µl of DPPH solution (each concentration was tested in 3 wells) and then incubated in a dark place for 30 minutes. The absorbance was measured at 517 nm using ELISA micro-plate reader (LabTech, Korea). A stock solution of each extract was prepared by dissolving (1mg/ml) in absolute methanol then diluted to the mentioned concentrations using absolute methanol as well. Vitamin C (ascorbic acid) was utilized as the positive control, absolute methanol alone was used as the blank, and 200 µl of DPPH solution plus 100 µl methanol was the negative control. The antioxidant activity (AA) percentage was calculated using the formula below [19]:

Where A_S, A_B, and A_C represent the absorbance of the test sample, blank, and control, respectively. 

Statistical analysis

Data were analyzed using the SPSS 16.0 and Microsoft Office Excel 2019. The results of (N = 8) are presented as means + SD and the comparison of the mean difference between groups was done by One-Way Analysis of Variance (ANOVA) test followed by Post-hoc Tukey test and the significance value was set at (P ≤ 0.05) for significant difference, and (P≤0.001 and 0.0001) for a highly significant difference. The concentration of the active extract that inhibited 50% of free radicals was determined according to the logarithmic equation by Microsoft Office Excel 2019.

Results and Discussion 

Extraction process

Powder of Olea europaea L. was extracted with three solvents (chloroform, methanol, and 50% ethanol) using the cold method (maceration) for extraction. The final yield weight of crude extract and percentage of 92 grams of the powder are shown in Table 1.

Biochemical analysis of liver indices

Liver indices were measured using serum levels of transaminases (ALT & AST) as well as ALP. Such biomarkers are useful for distinguishing early acute liver injury. Results of the study revealed a significant sharp elevation (P≤0.0001) in serum transaminases (ALT & AST) in the MTX group [receiving 20mg/kg MTX only as a single dose], suggesting a successful induction of acute liver injury when compared to the normal animal group. Animal group pretreated with silymarin served as animals receiving standard hepatoprotective agent, where the results revealed a significant reduction in serum values of transaminases (ALT & AST) (P≤0.001) when compared to the MTX group. Animal groups pretreated with Olea europaea extracts (group 4, 5, and 6) receiving Olea europaea chloroform extract, methanol extract and 50% ethanol extract respectively at 500mg/kg once daily dosing showed a statistically significant reduction in serum levels of transaminases (ALT & AST), (P≤0.05) for OE-CE and (P≤0.0001) for OE-ME and OE-EE when compared to MTX group as shown in Table 2 and Figure 1. Although, animals pretreated with 50% ethanol extract of Olea europaea (OE-EE) showed a better reduction in liver indices when compared to OE-CE and OE-ME pretreated groups (P≤0.05). Serum alkaline phosphatase (ALP) remained within normal levels among all animal groups; although the MTX group showed a mild elevation in ALP value, these values remained within normal intervals [20].

As for serum total protein (STP) and serum total bilirubin (TSB), the results of the experiment revealed that their values among all animal groups remained within normal intervals. Although; regarding STB, the MTX group showed a statistically significant increase in STB (P≤0.05) when compared to the normal group, still that elevation remained within normal intervals. As well as animals pretreated with (OE-EE) revealed a statistically significant reduction in STB (P≤0.001) when compared to the MTX group and the others pretreated groups as shown in Figure 2. Data analysis of STP revealed no significant difference among all animal groups (P = 0.46) as shown in Figure 3, and the values of TSP for all animal groups remained within normal intervals [20].

Results are expressed as mean + SD, (n = 8), the significance level was set at P≤0.05.

** represents highly significant difference (P≤0.0001)

GPT(ALT)= glutamic pyruvic transaminase (Alanine Transaminase), GOT (AST)= glutamic oxaloacetic transaminase (Aspartate Transaminase), ALP= Alkaline Phosphatase, TSB= Total Serum Bilirubin, TSP= Total Serum Protein, MTX= methotrexate, OE-CE= Olea europaea chloroform extract, OE-EE= Olea europaea 50% ethanol extract, OE-ME= Olea europaea methanol extract.

Histopathology description

The results of the examination revealed; liver section of the normal group showing the normal histological structure, which consists of central vein and arrangement of hepatocyte cells as a thread like in between sinusoid. The liver section of the animal group that received MTX only revealed a focal necrotic area with an accumulation of inflammatory cells with necrosis of hepatocyte cells, as well as accumulation of fat droplets and apoptotic cells are present around central veins. Histological liver section of animals pretreated with standard silymarin showing focal necrosis of hepatocytes with infiltration of inflammatory cells around the central vein. As for liver sections from animals pretreated with olive seeds (Olea europaea) extracts revealed varying results; animals pretreated with chloroform extract (OE-CE) showed marked inflammatory infiltrate, necrosis of hepatocytes and abundant accumulation of glycoprotein granules and dilated sinusoids. While in animals pretreated with methanol extract (OE-ME), the liver section revealed focal necrosis of hepatocytes around congested central vein but to a lesser extent with fewer inflammatory infiltration. Animals pretreated with 50% methanol extract (OE-EE) revealed the best results where the histological section showing a near-normal structure with a mild accumulation of lipid materials, these results are properly explained in (Figure 4). Stained liver sections were viewed under a light microscope with 40X, and the histological results obtained solidify and support the results of the biochemical analysis of liver indices (Figure 1).

Determination of free radical scavenging activity

Free radical scavenging (antioxidant) activity was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Each extract of Olea europaea L. seeds was tested with the use of a standard solution of DPPH free radical to observe the ability of each extract in reducing the free radical. Result of the study revealed that, all the extracts were able to scavenge DPPH free radical when they were compared to the negative control (P≤0.05). although, 50% ethanol extract of Olea europaea L. seeds (OE-EE) exhibited the best free radical scavenging activity compared to the negative control and the other extracts (P≤0.05) as shown in (Figure 5) with IC₅₀ of (5.48 µg/ml) according to the logarithmic equation. This could support the results obtained from the biochemical analysis of liver indices and the histological examination of liver tissues that 50% ethanol extract of Olea europaea L. seeds revealed a high statistical reduction in the levels of liver indices, as well as better histological results of livers tissue when compared to the other animal groups (P≤0.0001).